面包乳杆菌广谱细菌素BM173的生物合成机理研究

Bacteriocin produced by Lactic Acid Bacteria (LAB) can inhibit the growth of food spoilage bacteria and foodborne pathogenic bacteria, and has broad prospects for commercial application in the field of food safety. BM173 is a novel leaderless bacteriocin produced by Lactobacillus crustorum MN047, it has a broad target spectrum. However, the biosynthesic mechanism of BM173 is still unexplored. It has been recently discovered that there are antibiotic resistance-related gene (LmrB), drug efflux pump gene (DHA2) and antibiotic resistance regulation gene (MarR) at the upstream or downstream of the bacteriocin-encoding gene. Among them, LmrB is able to provide immunity to the host strain against BM173, and the expression of MarR and DHA2 is affected by the concentration of BM173. Thus, it is speculated that Lactobacillus crustorum may use those antibiotic resistance-related genes to regulate, transport, and immunize its own bacteriocin. This project uses molecular biology techniques including RT-PCR，RACE，EMSA，genome editing and protein labeling to study the biosynthetic and regulatory mechanisms of the novel broad-spectrum bacteriocin BM173. The expected results will provide the theoretical basis for further clarifying the synthetic mechanism of other class II leaderless bacteriocins, which is of great significance for promoting the utilization of LAB-produced bacteriocins.

乳酸菌细菌素能够抑制食品腐败菌和食源性致病菌，在食品安全领域具有广阔应用前景。BM173是由面包乳杆菌MN047产生的具有广谱抗菌活性的无前导肽II型细菌素，但其生物合成机理尚不清楚。申请人发现，BM173编码基因的上下游存在抗生素抗性基因（LmrB）、药物外排泵基因（DHA2）和耐药调节基因（MarR）。其中，LmrB能够为宿主提供针对BM173的免疫保护，MarR和DHA2的表达受到BM173浓度的影响。据此推测，三个抗生素抗性相关基因参与了BM173的生物合成过程。本项目通过RT-PCR、RACE、EMSA、基因编辑、蛋白标记等分子生物学技术，重点研究抗生素抗性机制在BM173表达调控、跨膜转运以及免疫等生物合成过程中的作用。项目的预期研究成果将为深入阐明无前导细菌素的合成机理提供理论依据，对促进乳酸菌细菌素的开发利用具有重要意义。

本项目对面包乳杆菌MN047所产新型II型细菌素BM173的合成调控机制进行了详细研究。项目首先开发了面包乳杆菌MN047的高效电转化方法，构建了面包乳杆菌MN047的基因编辑平台。利用此系统对10个细菌素编码基因，两个转录因子，以及乙醇脱氢酶基因的敲除效率明显提高，对一些基因的敲除效率高达100%。研究了面包乳杆菌细菌素的生物合成调控规律。总体来说，在高营养培养基如BHI，GM17中，细菌素表达强度明显弱于MRS培养基。在25°C，30°C以及37°C培养温度下，较高温度有利于细菌素的表达。培养pH对细菌素基因表达也有影响，在弱酸性（pH5.5-6.0）条件下，细菌素表达强度最强。培养基中的碳源（蔗糖）在0.1M是，细菌素基因表达强度最高。此外，研究了LuxS对细菌素表达的影响。发现LuxS主要通过影响群体感应系统，调控细菌素的表达。